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Reduced Representation Bisulfite Sequencing in Maize

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Epigenetics & Chromatin
Oct 2013



DNA methylation is an epigenetic modification that regulates plant development (Law and Jacobsen, 2010). Whole genome bisulfite sequencing (WGBS) is a state-of-the-art method for profiling genome-wide methylation patterns with single-base resolution (Cokus et al., 2008). However, for an organism with a large genome, e.g., the 2.1 Gb genome of maize, WGBS may be very expensive. Reduced representation bisulfite sequencing (RRBS) has been developed in mammalian studies (Smith et al., 2009). By digesting the genome with MspI with a size selection range of approximately 40-220 bp, CG-rich regions covering only ~1% of the human genome can be specifically sequenced. However, unlike mammalian genomes, plant genomes do not exhibit clear CpG islands. Therefore the original RRBS protocol is not suitable for plants. Accordingly, we developed an in silico pipeline to select specific enzymes to generate a region of interest (ROI)-enriched, e.g., promoter-enriched, reduced representation genome in plants (Hsu et al., 2017). By digesting the maize genome with MseI and selecting 40-300 bp segments, we sequenced about one-fourth of the maize genome while preserving 84.3% of the promoter information. The protocol has been successfully established in maize and can be broadly used in any genome. Our in silico pipeline is combined with the RRBS library preparation protocol, allowing for the computational analysis and experimental validation.

Keywords: Bisulfite sequencing (亚硫酸氢盐测序), DNA methylation ( DNA甲基化), Epigenetics (表观遗传学), Maize (玉米), Methylome (甲基化组)


DNA methylation is a heritable epigenetic modification that plays an important role in many developmental processes of animals, plants and fungi by regulating gene expression and the chromatin structure (Law and Jacobsen, 2010). WGBS is a genome-wide scale method for profiling DNA methylation at single-base resolution, although high sequencing costs are required to achieve sufficient coverage (Cokus et al., 2008). In mammals, RRBS has been developed to specifically sequence CG-dense regions, e.g., CpG islands (Smith et al., 2009). In this protocol, we aimed to adapt RRBS for plants. To be specific, we developed an in silico pipeline (Figure 1) to performed enzyme selection by targeting specific genome regions to generate RRBS methylomes and provided an experimental validation protocol.

Our method has been successfully established in the maize genome, one of the major global crops, which has a 2.1 Gb genome (Hsu et al., 2017). In addition to its large size, 85% of the maize genome consists of various repetitive sequences (Schnable et al., 2009). This feature could cause multiple mapping, i.e., many short reads from sequencing, which need to be discarded. These characteristics make targeted bisulfite sequencing (BS-seq) more cost-effective. mCHH islands were found to be located upstream of transcription start sites (TSSs) with higher methylation level (Gent et al., 2013; Li et al., 2015). We therefore aimed to perform promoter-enriched RRBS in maize, and an in silico pipeline was developed for enzyme selection.

Our in silico pipeline currently has 85 pre-installed restriction enzymes. Users can easily append more enzymes. A genome FASTA file, refFlat annotation file, and repeat gff3 annotation file are the required input files to run this pipeline. ROIs, including promoters, exons, introns, splicing sites, repeats, UTRs and intergenic regions are pre-selected for enrichment analysis. As soon as the input files are prepared, users can run our pipeline to select ideal enzymes by typing simple commands. Users can also verify the prediction by performing RRBS library construction following the experimental protocol provided and performing sequencing.

Figure 1. Flowchart of maize RRBS in silico pipeline

Materials and Reagents

  1. Pipette tips
    Note: Low retention tips are recommended.
  2. 1.5 ml microcentrifuge tubes (Eppendorf, catalog number: 0030108051 )
  3. PCR tubes (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: A30588 )
  4. Clean razor blade
  5. Qubit dsDNA BR Assay Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: Q32850 ) (used to quantify double strand DNA in combination with Qubit Fluorometer)
  6. MseI (New England Biolabs, catalog number: R0525S ) (used to digest maize genomic DNA, CutSmart Buffer is included)
    Note: The enzyme could be replaced according to the in silico enzyme selection result in Procedure A.
  7. Agarose
    Note: For the gel size selection, we recommend low melting point agarose.
  8. TBE buffer (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15581044 )
  9. AMPure XP (Beckman Coulter, catalog number: A63881 )
  10. Absolute ethanol
    Note: To dilute, use DNase/RNase-free distilled water.
  11. 1 M Tris-HCl (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15568025 )
    Note: To dilute, use DNase/RNase-free distilled water.
  12. NEBuffer 2 (New England Biolabs, catalog number: B7002S )
  13. Klenow Fragment (3’→5’ exo-) (New England Biolabs, catalog number: M0212S ) (used to end-repair and A-tail DNA)
  14. T4 DNA Ligase (New England Biolabs, catalog number: M0202S , T4 DNA ligation buffer is included) (used to ligate sequencing adaptors)
  15. 100-bp DNA ladder
  16. QIAquick Gel Extraction Kit (QIAGEN, catalog numbers: 28704 , 28706 )
  17. EpiTect Fast Bisulfite Conversion Kit (QIAGEN, catalog number: 59824 )
    Note: Keep this kit at an appropriate temperature according to the manufacturer’s guide.
  18. TruSeq DNA LT Sample Prep Kit (Illumina, catalog number: FC-121-2001 )
  19. PfuTurbo DNA Polymerase (Agilent Technologies, catalog number: 600250 )
  20. DNase/RNase-free distilled water (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10977015 )
  21. Deoxynucleotide (dNTP) Solution Set (New England Biolabs, catalog number: N0446S )
  22. MinElute PCR Purification Kit (QIAGEN , catalog number: 28004 )
  23. 10 mM dNTP mix (see Recipes)
  24. 5x RRBS dNTP mix (see Recipes)


  1. Pipettes:
    1. Thermo Fisher Scientific, Thermo ScientificTM, model: P5000, catalog number: 4641110N
    2. Thermo Fisher Scientific, Thermo ScientificTM, model: P1000, catalog number: 4641100N
    3. Thermo Fisher Scientific, Thermo ScientificTM, model: P300, catalog number: 4641090N
    4. Thermo Fisher Scientific, Thermo ScientificTM, model: P200, catalog number: 4641080N
    5. Thermo Fisher Scientific, Thermo ScientificTM, model: P100, catalog number: 4641070N
    6. Thermo Fisher Scientific, Thermo ScientificTM, model: P20, catalog number: 4641060N
    7. Thermo Fisher Scientific, Thermo ScientificTM, model: P10, catalog number: 4641030N
    8. Thermo Fisher Scientific, Thermo ScientificTM, model: P2, catalog number: 4641010N
  2. Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Invitrogen, model: Qubit® 2.0 Fluorometer , catalog number: Q32866)
  3. Heating block (Eppendorf, model: ThermoMixer comfort , catalog number: 5355000011)
  4. Incubator (Only Science, Firstek, model: S300 )
  5. Thermocycler
    1. Thermo Fisher Scientific, Applied BiosystemsTM, model: GeneAmpTM PCR System 9700 , catalog number: 4413750
    2. Bio-Rad Laboratories, model: T100TM Thermal Cycler, catalog number: 1861096
  6. Centrifuge
    1. Eppendorf, model: Centrifuge MiniSpin® plus , catalog number: 5453000011
    2. Eppendorf, model: Centrifuge 5424 , catalog number: 5424000410
    3. GYROZEN, model: 1730R
  7. Magnetic stand
    1. Thermo Fisher Scientific, DynaMagTM-2 Magnet, catalog number: 12321D
    2. Thermo Fisher Scientific, Applied BiosystemsTM, DynaMagTM-PCR Magnet, catalog number: 492025


  1. System requirements: Linux/Unix or Mac OS, python 2.7+, R 3.2.0
  2. Python and R scripts and a tutorial are available at: https://gitlab.com/fmhsu0114/maize_RRBS


The complete pipeline contains 1. in slico enzyme selection, followed by 2. RRBS library preparation. The resulted library is to be sequenced to generate DNA methylation profiles at genomic regions of interest.

  1. In silico enzyme selection
    1. Clone the GitLab project from: https://gitlab.com/fmhsu0114/maize_RRBS.
    2. Download the genome FASTA file of your interest, i.e., maize_genome.fa, and use the following command to build the genome database:
      $ python Build_Gnome.py AGPv3.fa
      2 output files (genome.shelve and log-genome.txt) will be generated.
    3. Download the annotation refFlat file, i.e., refFlat.txt, and use the following command to build the gene annotation database:
      $ python Build_refgene_shelve.py refFlat.txt log-genome.txt
      1 output file (refgenes.shelve) will be generated.
    4. Download the annotation gff3 file and grep ‘repeats’ into an individual file, i.e., repeats.gff3, and use the following command to build the repeat annotation database:
      $ python Build_repeats_Gnome.py repeats.gff3, log-genome.txt
      1 output file (repeat.shelve) will be generated.
    5. Build the genome CG, CHG and CHH sites database with the following command:
      $ python Build_CG_CHG_CHH_Gnome.py genome.shelve log-genome.txt
      1 output file (CG_CHG_CHH.shelve) will be generated.
    6. Perform in silico digestion with the provided enzyme list in the GitLab project (enzyme_cutting_sites.txt) with the following command:
      $ python Enzyme_digestione.py
      fragment_info (per MSRE and per size range) for each enzyme and a Summary-enzyme-coverage.txt file will be generated.
    7. Place the output files from 1.6 into one directory ./fragment/ and run the enrichment analysis with the following command:
      $ python RRBS_fragments_enrichment.py repeats.shelve, refgenes.shelve
      log2_fold_enrichment (per MSRE and per size range) files will be generated (see Table 1).

      Table 1. Fold enrichment values in log2 scale of the MseI 40-300-bp fragments

    8. Move all of the log2_fold_enrichment files into one directory ./output/ and plot the enrichment bar charts with the following command:
      $ Rscript Barchart_enrichment.R
      See Figure 2 to Figure 4 as examples.

      Figure 2. Bar chart of the MseI 40-300-bp fragments shows enrichment in promoters

      Figure 3. Bar chart of the CviQI 40-280-bp fragments shows enrichment in exons, introns, UTRs and splice sites

      Figure 4. Bar chart of the BfaI 50-300-bp fragments shows enrichment in promoters

    9. Use the following command to calculate the covered promoter and genebody region:
      $ python RRBS_fragments_enrichment_gene_level_per_MRE_Size.py -i Enzyme -s size_range -c 1
      This script has 3 parameters:
      -i: restriction enzyme
      -s: size selection range, e.g., 40-280
      -c: number of cytosines per promoter/genebody to be included
      An output file, Fragment-Enrichment-genebody-pmt-Enzyme-size_range-1.txt, will be generated (Table 2 and Table 3). This file contains the percentages of the promoters and genebody covered in the reduced representation (RR) genome.
      Note: In Table 2 and Table 3, Pct refers to percentage; Pmt refers to promoter.

      Table 2. Promoter and genebody covered in the MseI 40-300-bp fragments

      Table 3. Promoter and genebody covered in the BfaI 50-300-bp fragments

  2. RRBS library preparation
    1. Use Qubit Fluorometric Quantification with Qubit dsDNA BR assay to quantify maize genomic DNA according to the manufacturer’s instructions.
      Note: To acquire high quality double strand genomic DNA, we recommend spin column-based DNA extraction kit, e.g., QIAGEN DNeasy Plant Mini kit. If Pheno-Chloroform method is used, we suggest another round of spin-column based purification.
    2. Aliquot 1 μg of maize genomic DNA into a 1.5 ml tube, add 5 μl of 10x CutSmart Buffer, 1 μl of MseI and ddH2O to a final volume of 50 μl. Mix carefully with a pipette and incubate at 37 °C on the heating block for 4 h.
      Note: MseI used here is designed for promoter-enriched RRBS in maize. For different genomes (organisms), researchers should conduct ‘in silico enzyme selection’ and replace MseI with the predicted enzyme, and modify the digestion time, buffer and temperature according to the manufacturer’s instructions.
    3. [Optional] Take 5 μl for gel electrophoresis (100 ng, 1.5%, 0.5x TBE) and add 5 μl ddH2O to the enzyme digestion product.
      Note: This step is to confirm the enzyme digestion efficiency.
    4. Post-digestion AMPure beads purification
      1. Add 150 μl of well-inverted AMPure XP beads solution to 50 μl of DNA, vortex for 10 sec, briefly centrifuge and incubate at RT for 5 min.
        Note: When vortexing solutions with AMPure XP beads, avoid high vortexing strength causing bubbles. After vortexing, use microcentrifuge to spin down quickly to remove liquids from the lid and the wall of the tube.
      2. Place the tube on the magnetic stand for 3 min.
      3. Remove the supernatant.
      4. Add 200 μl of 80% EtOH without disturbing the beads and incubate at RT for 30 sec. Remove the EtOH.
        Note: Do not remove the tube from the magnetic stand.
      5. Repeat the washing Step B4d.
      6. Air-dry the beads for 5 min.
      7. Remove the tube from the magnetic stand.
      8. Add 32 μl of 10 mM Tris-HCl and pipet up and down to mix thoroughly. Incubate at RT for 2 min.
      9. Place the tube on the magnetic stand for 3 min.
      10. Transfer the supernatant to a PCR tube.
    5. End repair and A-tailing
      1. Add 6 μl of 10x NEBuffer 2, 12 μl of 5x RRBS dNTP mix and 3 μl of 3’→5’ Klenow exo- to the purified DNA and add ddH2O to a final volume of 60 μl.
      2. Incubate using thermocycler at 30 °C for 20 min and 37 °C for 20 min, then place the tube on ice.
    6. DNA purification with AMPure XP beads
      1. Add 180 μl of well-inverted AMPure XP beads to the 60 μl of DNA, vortex for 10 sec, briefly centrifuge and incubate at RT for 5 min.
      2. Place the tube on the magnetic stand for 3 min.
      3. Remove the supernatant.
      4. Add 200 μl of 80% EtOH without disturbing the beads and incubate at RT for 30 sec. Remove the EtOH.
        Note: Do not remove the tube from the magnetic stand.
      5. Repeat the washing Step B6d.
      6. Air-dry the beads for 5 min.
      7. Remove the tube from the magnetic stand.
      8. Add 24 μl of 10 mM Tris-HCl and pipet up and down to mix thoroughly. Incubate at RT for 2 min.
      9. Place the tube on the magnetic stand for 3 min.
      10. Transfer the supernatant to a PCR tube.
    7. Ligate with a TruSeq methylated Illumina adaptor
      1. Add 3 μl of 10x T4 DNA ligation buffer, 2 μl of adaptor and 1 μl of T4 DNA ligase to the 24 μl end-repaired and A-tailed DNA to a final volume of 30 μl.
      2. Incubate at 16 °C overnight in a thermocycler.
    8. Gel size selection (Figure 5)
      1. Load all the adapter-ligated DNA to 1.5% 0.5x TBE gel (15 μl per well, in total 2 wells) for gel electrophoresis with a 100-bp DNA ladder.
      2. Use a clean razor blade for each sample to cut 40-300-bp fragments and purify the DNA using the QIAGEN Qiaquick Gel Extraction Kit following the manufacturer’s instructions. Elute DNA twice with a total volume of 34 μl of EB buffer. Transfer the solution to a PCR tube. Take 1 μl for Qubit quantification (optional).
      Note: One should avoid UV light during gel size selection. Since the adaptor sequence is about 120 bp, we cut 160-420-bp fragments from the gel. For each well in the previous step, use one QIAGEN Qiaquick Gel Extraction reaction. For the first round of elution, add 10 μl of EB buffer to each MinElute spin column, and collect the 20 μl of the solution in a new tube. Then use another 14 μl of EB buffer to elute the remaining DNA from each spin column one by one.

      Figure 5. Gel size selection

    9. Bisulfite conversion
      Note: Confirm that the reagents in the EpiTect Fast Bisulfite Conversion Kit are ready to use.
      1. Add RNase-free water to the DNA solution to a final volume of 40 μl, mix with a pipette and aliquot 20 μl to another PCR tube.
      2. Add 35 μl of DNA Protect Buffer and 85 μl of a Bisulfite Solution to each tube to a final volume of 140 μl and mix thoroughly with gentle pipetting.
      3. Run in a thermocycler with the following program:

        Then hold at 20 °C. Lid temperature: 100 °C.
    10. Briefly centrifuge the PCR tubes containing the bisulfite reaction with microcentrifuge to remove the liquid from the tube lid and transfer to clean 1.5 ml microcentrifuge tubes. For each tube, add 310 μl of freshly prepared Buffer BL containing 10 μg/ml carrier RNA, mix thoroughly with pipetting and then centrifuge briefly.
    11. Add 250 μl of 96-100% EtOH, gently pipet for 10 sec and then centrifuge briefly.
    12. Transfer all of the solution to MinElute DNA spin columns with collection tubes. Centrifuge at maximum speed for 1 min and discard the flow-through.
    13. Washing
      Add 500 μl Buffer BW to each spin column, centrifuge at maximum speed for 1 min and discard the flow-through.
    14. Desulfonation
      1. Add 500 μl of Buffer BD to each spin column and incubate for 15 min at RT.
      2. Centrifuge at maximum speed for 1 min and discard the flow-through.
      3. Add 500 μl Buffer BW to each spin column, centrifuge at maximum speed for 1 min and discard the flow-through.
      4. Repeat the washing Step B14c.
      5. Add 250 μl of 96-100% EtOH to each spin column and centrifuge at maximum speed for 1 min.
      6. Place each spin column into a clean 1.5 ml microcentrifuge tube, open the lid and incubate using a 60 °C heating block to evaporate the liquid.
      7. Elute the DNA by adding 15 μl of Buffer EB onto the center of the spin column membrane, close the lid and incubate at RT for 1 min.
      8. Centrifuge at 15,000 x g for 1 min.
    15. PCR amplification
      1. Add 2.5 μl Illumina TruSeq PCR primer cocktail, 0.5 μl of 10 mM dNTP, 5 μl of 10x Pfu Turbo Buffer, 0.5 μl of Pfu Turbo DNA polymerase and 26.5 μl of ddH2O to the eluted DNA solution to a final volume of 50 μl. Mix thoroughly and centrifuge briefly.
      2. Run in a thermocycler with the following program:

    16. DNA purification with AMPure XP beads
      1. Add 150 μl of well-inverted AMPure XP beads to the 50 μl of DNA, vortex for 10 sec, briefly centrifuge and incubate at RT for 5 min.
      2. Place the tube on the magnetic stand for 3 min.
      3. Remove the supernatant.
      4. Add 200 μl of 80% EtOH without disturbing the beads and incubate at RT for 30 sec. Remove the EtOH.
        Note: Do not remove the tube from the magnetic stand.
      5. Repeat the washing Step B16d.
      6. Air-dry the beads for 5 min.
      7. Remove the tube from the magnetic stand.
      8. Add 22 μl of 10 mM Tris-HCl and pipet up and down to mix thoroughly. Incubate at RT for 2 min.
      9. Place the tube on the magnetic stand for 3 min.
      10. Transfer the supernatant to a 1.5 ml microcentrifuge tube.
    17. Quantify the final library concentration with the Qubit assay.
    18. Sequence the RRBS library with Illumina paired-end sequencing. Any regular length of pair-end reads, e.g., 100-250 bp, is fine.

Data analysis

  1. Our in silico pipeline creates a table and bar chart for each enzyme with its associated range of size selection. Figure 1 is a flowchart of this pipeline, and users could see input and output files of each step. Figure 2 is a bar chart showing the enrichment of each potential ROI from approximately 40-300 bp size-selected MseI-digested DNA. The X-axis is in log2 scale, so values > 0 can be considered to be an enrichment of these MseI-digested DNA fragments. Table 1 includes the values used to plot Figure 2. From Figure 2 and Table 1, we can see that the MseI-digested 40-300-bp fragments are enriched in promoters. As an alternative, Figure 3 shows the result from another enzyme (CviQI) 40-280 bp, with fragments more enriched in exons, splice sites, introns and UTRs.
  2. Different enzymes produce similar enrichment values. Figure 2 and Figure 4 suggest that MseI 40-300-bp and BfaI 50-300-bp fragments are enriched in promoters. Table 2 and Table 3 show the statistics of the promoters and genebody percentages covered. For the MseI RR genome, by sequencing 566 Mbp of the 2.1 Gb genome, 84.3% of promoters are included. As for the BfaI RR genome, sequencing 23.0% of the genome includes 74.8% of the promoters. Users need to determine the tradeoff between the cost of 4.4% of the genome and 8.5% of the promoters.


  1. We present an in silico pipeline to perform ROI-directed RRBS in maize with an experimentally verifiable prediction. The most critical steps for users to run our pipeline are: 1) Preparing the input files; 2) Choosing an optimal enzyme to show clear enrichment of the selected ROIs (see Figure 2).
  2. Input files can often be downloaded from public databases, such as Ensembl (Zerbino et al., 2017). It is important to note that our pipeline requires an individual repeat annotation file in .gff3 format. In addition to the bar charts and table (Figures 2 and 3, Table 1), when judging which size-selection region is optimal, the total RR genome size and read length of the sequencing platform should also be considered. For example, if two RR genomes show 2x enrichment in promoters and one covers 50% of the whole genome while the other covers 25%, the tradeoff between sequencing cost and quantity of information should be considered.
  3. For experimental validation, size selection and bisulfite conversion are the most critical steps. In this protocol, we used gel size selection to ensure that the fragment length could be visualized. As an alternative, AMPure XP beads can also be used. To ensure efficient bisulfite conversion, 0.01% sheared lambda phage DNA could be pooled into original genomic DNA prior to enzyme digestion. Since lambda phage DNA lacks methylated cytosine, non-conversion rate can be estimated by calculating the methylation level of lambda phage DNA to evaluate bisulfite conversion efficiency.


  1. 10 mM dNTP mix
    10 mM dATP
    10 mM dTTP
    10 mM dCTP
    10 mM dGTP
  2. 5x RRBS dNTP mix
    200 μM dATP
    200 μM dTTP


This study was supported by funding from the Institute of Plant and Microbial Biology, Academia Sinica and grants obtained from Taiwan Ministry of Science and Technology (106-2633-B-001-001) to P.-Y.C. We thank Yi-Jing Lee for providing the equipment information. The authors declare that they have no competing financial interests.


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  7. Smith, Z. D., Gu, H., Bock, C., Gnirke, A. and Meissner, A. (2009). High-throughput bisulfite sequencing in mammalian genomes. Methods 48(3): 226-232.
  8. Zerbino, D. R., Achuthan, P., Akanni, W., Amode, M. R., Barrell, D., Bhai, J., Billis, K., Cummins, C., Gall, A., Giron, C. G., Gil, L., Gordon, L., Haggerty, L., Haskell, E., Hourlier, T., Izuogu, O. G., Janacek, S. H., Juettemann, T., To, J. K., Laird, M. R., Lavidas, I., Liu, Z., Loveland, J. E., Maurel, T., McLaren, W., Moore, B., Mudge, J., Murphy, D. N., Newman, V., Nuhn, M., Ogeh, D., Ong, C. K., Parker, A., Patricio, M., Riat, H. S., Schuilenburg, H., Sheppard, D., Sparrow, H., Taylor, K., Thormann, A., Vullo, A., Walts, B., Zadissa, A., Frankish, A., Hunt, S. E., Kostadima, M., Langridge, N., Martin, F. J., Muffato, M., Perry, E., Ruffier, M., Staines, D. M., Trevanion, S. J., Aken, B. L., Cunningham, F., Yates, A. and Flicek, P. (2017). Ensembl 2018. Nucleic Acids Res.


DNA甲基化是调节植物发育的表观遗传修饰(Law and Jacobsen,2010)。全基因组亚硫酸氢盐测序(WGBS)是用单碱基分辨率分析全基因组甲基化模式的最先进的方法(Cokus et al。,2008)。然而,对于具有大基因组的生物体,例如玉米的2.1Gb基因组,WGBS可能非常昂贵。代表性亚硫酸氢盐测序(RRBS)已经在哺乳动物研究中发展(Smith等人,2009)。通过用大小选择范围大约40-220bp的 Msp 消化基因组,可以对仅涵盖〜1%人类基因组的CG富含区域进行特异性测序。然而,与哺乳动物基因组不同,植物基因组不显示清楚的CpG岛。因此原来的RRBS协议不适用于工厂。因此,我们开发了一种计算机管道来选择特定的酶以生成感兴趣区域(ROI) - 富集的,例如,富含启动子的,减少的植物表达基因组(例如, Hsu et al。,2017)。通过用MseI消化玉米基因组并选择40-300bp片段,我们测序了大约四分之一的玉米基因组,同时保留了84.3%的启动子信息。该协议已在玉米中成功建立,可广泛应用于任何基因组。我们的计算机管道系统与RRBS文库制备方案相结合,允许进行计算分析和实验验证。

【背景】DNA甲基化是一种可遗传的表观遗传修饰,通过调节基因表达和染色质结构在动物,植物和真菌的许多发育过程中发挥重要作用(Law and Jacobsen,2010)。 WGBS是一种全基因组范围的方法,用于在单碱基分辨率下分析DNA甲基化,尽管需要较高的测序成本才能获得足够的覆盖率(Cokus et al。,2008)。在哺乳动物中,RRBS已被开发用于特异性地测序CG-密集区域,例如CpG岛(Smith等人,2009)。在这个协议中,我们的目标是使RRBS适用于植物。具体而言,我们开发了一种计算机管道(图1),通过靶向特定的基因组区域来产生RRBS甲基化组织并提供实验验证方案,从而进行酶选择。

我们的方法已经在玉米基因组中成功建立,玉米基因组是全球主要作物之一,它拥有2.1 Gb基因组(Hsu et al。,2017)。除了它的大尺寸之外,85%的玉米基因组由各种重复序列组成(Schnable et al。 ,2009)。此功能可能会导致多次映射,即 ,因此需要丢弃许多来自排序的简短读取。这些特性使靶向亚硫酸氢盐测序(BS-seq)更具成本效益。发现mCHH岛位于具有较高甲基化水平的转录起始位点(TSS)的上游(Gent et al。,2013; Li等人,2015)。因此,我们的目标是在玉米中执行富含启动子的RRBS,并开发用于酶选择的计算机管线。

我们的电脑管道目前有85个预装的限制性内切酶。用户可以轻松追加更多的酶。基因组FASTA文件,refFlat注释文件和repeat gff3注释文件是运行此管道所需的输入文件。 ROI包括启动子,外显子,内含子,剪接位点,重复序列,非编码区和基因间区域都被预先选定用于富集分析。只要准备好输入文件,用户就可以运行我们的管道,通过输入简单命令来选择理想的酶。用户还可以按照所提供的实验方案执行RRBS库构建并执行测序来验证预测结果。

图1.玉米RRBS in silico 管道的流程图

关键字:亚硫酸氢盐测序, DNA甲基化, 表观遗传学, 玉米, 甲基化组


  1. 移液器吸头
  2. 1.5 ml微量离心管(Eppendorf,目录号:0030108051)
  3. PCR管(Thermo Fisher Scientific,Applied Biosystems TM,目录号:A30588)。
  4. 清洁剃须刀片
  5. Qubit dsDNA BR Assay Kit(Thermo Fisher Scientific,Invitrogen TM,目录号:Q32850)(用于量化与Qubit荧光计结合的双链DNA)
  6. (新英格兰生物实验室,目录号:R0525S)(用于消化玉米基因组DNA,包括CutSmart缓冲液)
  7. 琼脂糖
  8. TBE缓冲液(Thermo Fisher Scientific,Invitrogen TM,目录号:15581044)
  9. AMPure XP(Beckman Coulter,目录号:A63881)
  10. 绝对乙醇
    注意:要稀释,请使用不含DNase / RNase的蒸馏水。
  11. 1M Tris-HCl(Thermo Fisher Scientific,Invitrogen TM,目录号:15568025)
    注意:要稀释,请使用不含DNase / RNase的蒸馏水。
  12. NEBuffer 2(New England Biolabs,目录号:B7002S)
  13. Klenow片段(3'→5'exo-)(New England Biolabs,目录号:M0212S)(用于末端修复和A尾DNA)
  14. T4 DNA连接酶(New England Biolabs,目录号:M0202S,包括T4 DNA连接缓冲液)(用于连接测序接头)
  15. 100-bp DNA梯子
  16. QIAquick凝胶提取试剂盒(QIAGEN,产品目录号:28704,28706)
  17. EpiTect快速亚硫酸氢盐转化试剂盒(QIAGEN,目录号:59824)
  18. TruSeq DNA LT样品制备试剂盒(Illumina,目录号:FC-121-2001)
  19. PfuTurbo DNA聚合酶(Agilent Technologies,目录号:600250)
  20. 不含DNase / RNase的蒸馏水(Thermo Fisher Scientific,Invitrogen TM,目录号:10977015)
  21. 脱氧核苷酸(dNTP)溶液组(New England Biolabs,目录号:N0446S)
  22. MinElute PCR纯化试剂盒(QIAGEN,目录号:28004)
  23. 10 mM dNTP混合物(见食谱)
  24. 5倍RRBS dNTP混合(见食谱)


  1. 移液器:
    1. Thermo Scientific Scientific,Thermo Scientific TM ,型号:P5000,目录号:4641110N
    2. Thermo Scientific Scientific,Thermo Scientific TM ,型号:P1000,目录号:4641100N
    3. 赛默飞世尔科技有限公司 TM ,型号:P300,目录号:4641090N
    4. Thermo Scientific Scientific,Thermo Scientific TM,型号:P200,目录号:4641080N
    5. 赛默飞世尔科技Thermo Scientific TM ,型号:P100,目录号:4641070N
    6. 赛默飞世尔科技Thermo Scientific TM ,型号:P20,目录号:4641060N
    7. 赛默飞世尔科技Thermo Scientific TM ,型号:P10,目录号:4641030N
    8. Thermo Scientific Scientific,Thermo Scientific T M,型号:P2,目录号:4641010N
  2. Qubit 2.0荧光计(Thermo Fisher Scientific,Invitrogen,型号:Qubit 2.0荧光计,目录号:Q32866)
  3. 加热块(Eppendorf,型号:ThermoMixer comfort,目录号:5355000011)
  4. 孵化器(仅科学,Firstek,型号:S300)
  5. 热循环仪
    1. Thermo Fisher Scientific,Applied Biosystems TM,型号:GeneAmp TM PCR System 9700,目录号:4413750
    2. Bio-Rad Laboratories,型号:T100 TM Thermal Cycler,产品目录号:1861096
  6. 离心分离机
    1. Eppendorf,型号:Centrifuge MiniSpin ®,产品目录号:5453000011
    2. Eppendorf,型号:离心机5424,目录号:5424000410
    3. GYROZEN,型号:1730R
  7. 磁性支架
    1. Thermo Fisher Scientific,DynaMag TM -2磁铁,目录号:12321D
    2. Thermo Fisher Scientific,Applied Biosystems TM,DynaMag TM -PCR Magnet,目录号:492025


  1. 系统要求:Linux / Unix或Mac OS,Python 2.7+,R 3.2.0
  2. Python和R脚本和教程可在以下网址找到: https://gitlab.com/fmhsu0114/maize_RRBS < br />


完整的流水线包含1. in slico 酶选择,然后是2. RRBS文库制备。所产生的文库将被测序以在感兴趣的基因组区域产生DNA甲基化概况。

  1. 在计算机中选择酶
    1. 克隆GitLab项目: https://gitlab.com/fmhsu0114/maize_RRBS
    2. 下载您感兴趣的基因组FASTA文件,即 ,maize_genome.fa,然后使用以下命令构建基因组数据库:
      $ python Build_Gnome.py AGPv3.fa
    3. 下载注释refFlat文件,即,refFlat.txt,然后使用以下命令构建基因注释数据库:
      $ python Build_refgene_shelve.py refFlat.txt log-genome.txt
    4. 将注释gff3文件和grep'repeats'下载到单个文件中,即,repeats.gff3,然后使用以下命令构建重复注释数据库:
      $ python Build_repeats_Gnome.py repeats.gff3,log-genome.txt
    5. 使用以下命令构建基因组CG,CHG和CHH站点数据库:
      $ python Build_CG_CHG_CHH_Gnome.py genome.shelve log-genome.txt
    6. 使用以下命令在GitLab项目(enzyme_cutting_sites.txt)中提供的酶列表执行 in silico 消化:
      $ python Enzyme_digestione.py
    7. 将输出文件从1.6放到一个目录./fragment/中,并使用以下命令运行浓缩分析:
      $ python RRBS_fragments_enrichment.py repeats.shelve,refgenes.shelve

      表1. Mse I 40-300-bp片段的log2浓度倍数富集值

    8. 将所有log 2 _fold_enrichment文件移动到一个目录中./output/并使用以下命令绘制浓缩条形图:
      $ Rscript Barchart_enrichment.R


      图2. I 40-300-bp片段的条形图显示了启动子的富集

      图3. Cvi的条形图QI 40-280-bp片段显示富含外显子,内含子,UTR和剪接位点 >

      图4. BFA的条形图I 50-300-bp片段显示了启动子的富集

    9. 使用以下命令来计算覆盖的启动子和基因体区域:
      $ python RRBS_fragments_enrichment_gene_level_per_MRE_Size.py -i Enzyme -s size_range -c 1
      注:在表2和表3中,Pct指的是百分比; Pmt是指启动子。

      表2. 40-300-bp片段中包含的启动子和基因体

      表3.在 Bfa I 50-300-bp片段中包含的启动子和基因体

  2. RRBS图书馆准备
    1. 根据制造商的说明,使用Qubit荧光定量法与Qubit dsDNA BR分析来定量玉米基因组DNA。
      注意:为了获得高质量的双链基因组DNA,我们推荐基于旋转柱的DNA提取试剂盒,例如QIAGEN DNeasy Plant Mini试剂盒。如果使用Pheno-Chloroform方法,我们建议再进行一次基于离心柱的纯化。
    2. 将1微克玉米基因组DNA分装入1.5毫升试管中,加入5微升10x CutSmart缓冲液,1微升MseI I和ddH 2 O至终体积为50微升。
    3. [可选]取5μl用于凝胶电泳(100 ng,1.5%,0.5x TBE),并向酶消化产物中加入5μlddH 2 O。
    4. 后消化AMPure珠子纯化
      1. 加入150μl反相AMPure XP珠溶液到50μlDNA中,涡旋10秒,短暂离心并在室温孵育5分钟。
        注意:当使用AMPure XP珠粒涡旋溶液时,避免高涡旋强度导致气泡。涡旋振荡后,使用微量离心机快速旋转以去除盖子和管壁上的液体。
      2. 将试管放在磁力架上3分钟。
      3. 去除上清液。
      4. 加入200μl的80%乙醇,不打扰珠子,并在室温孵育30秒。去除EtOH。
      5. 重复洗涤步骤B4d。
      6. 将珠子风干5分钟。
      7. 从磁力架上取下管子。
      8. 加入32μl10mM Tris-HCl并上下移液以充分混合。在室温孵育2分钟。
      9. 将试管放在磁力架上3分钟。
      10. 将上清液转移到PCR管中。
    5. 结束修理和A-tailing
      1. 向纯化的DNA中加入6μl10x NEBuffer2,12μl5×RRBS dNTP混合物和3μl3'→5'Klenow外切酶并加入ddH 2 O至终体积60μl 。
      2. 使用热循环仪在30°C孵育20分钟和37°C孵育20分钟,然后将试管置于冰上。
    6. 用AMPure XP珠子进行DNA纯化
      1. 将180μl反相良好的AMPure XP珠加入到60μlDNA中,涡旋10秒,短暂离心并在室温孵育5分钟。
      2. 将试管放在磁力架上3分钟。
      3. 去除上清液。
      4. 加入200μl的80%乙醇,不打扰珠子,并在室温孵育30秒。去除EtOH。
      5. 重复洗涤步骤B6d。
      6. 将珠子风干5分钟。
      7. 从磁力架上取下管子。
      8. 加入24μl的10 mM Tris-HCl并上下吸取混匀。在室温孵育2分钟。
      9. 将试管放在磁力架上3分钟。
      10. 将上清液转移到PCR管中。
    7. 用TruSeq甲基化Illumina适配器进行连接
      1. 加入3微升10x T4 DNA连接缓冲液,2微升适配器和1微升T4 DNA连接酶到24微升末端修复和A尾巴的DNA,最终体积为30微升。

      2. 在16°C孵育过夜的热循环仪
    8. 凝胶大小选择(图5)
      1. 将所有接头连接的DNA加载到1.5%0.5x TBE凝胶(每孔15μl,总共2个孔)中,用100-bp DNA梯进行凝胶电泳。
      2. 每个样品使用干净的剃须刀片切割40-300-bp片段,并使用QIAGEN Qiaquick凝胶提取试剂盒按照生产商的说明纯化DNA。用总体积为34μl的EB缓冲液洗脱DNA两次。将溶液转移到PCR管中。取1μl用于Qubit定量(可选)。
      注意:在选择凝胶尺寸时应避免紫外线。由于接头序列大约为120bp,因此我们从凝胶中切下了160-420bp的片段。对于上一步中的每个孔,请使用一个QIAGEN Qiaquick Gel Extraction反应。对于第一轮洗脱,向每个MinElute离心柱中加入10μlEB缓冲液,并将20μl溶液收集到一个新管中。然后使用另外14微升EB缓冲液逐一洗脱每个离心柱中剩余的DNA。


    9. 亚硫酸氢盐转化
      注意:确认EpiTect Fast Bisulfite转换工具包中的试剂已准备好使用。
      1. 向DNA溶液中加入不含RNase的水至终体积为40μl,用移液管混匀并将20μl等分至另一个PCR管中。
      2. 加入35μl的DNA保护缓冲液和85μl的亚硫酸氢盐溶液到每个管中,最终体积为140μl,并用轻柔移液彻底混合。
      3. 使用以下程序在热循环仪中运行:

    10. 用微量离心机轻轻离心含有亚硫酸氢盐反应的PCR管,以从管盖中取出液体并转移到清洁的1.5ml微量离心管中。对于每个试管,加入310μl含有10μg/ ml载体RNA的新鲜制备的缓冲液BL,用移液器充分混合,然后短暂离心。
    11. 加入250μl的96-100%乙醇,轻轻吸取10秒,然后短暂离心。
    12. 将所有的解决方案都转移到带有收集管的MinElute DNA离心柱上。以最大速度离心1分钟并丢弃流通液。
    13. 洗涤
    14. 脱磺化
      1. 向每个离心柱加入500μl缓冲液BD,并在室温孵育15分钟。

      2. 最大速度离心1分钟并丢弃流通液。
      3. 向每个离心柱加入500μl缓冲液BW,以最大速度离心1分钟并丢弃流出液。
      4. 重复洗涤步骤B14c。
      5. 向每个离心柱中加入250μl96-100%EtOH,并以最大速度离心1分钟。
      6. 将每个离心柱放入干净的1.5 ml微量离心管中,打开盖子并使用60°C加热块进行孵育以蒸发液体。
      7. 通过向离心柱膜的中心添加15μl缓冲液EB来洗脱DNA,盖上盖子并在室温孵育1分钟。

      8. 在15,000×g g离心1分钟。
    15. PCR扩增
      1. 向洗脱的DNA溶液中加入2.5μlIllumina TruSeq PCR引物混合物,0.5μl10mM dNTP,5μl10x Pfu Turbo缓冲液,0.5μlPfu Turbo DNA聚合酶和26.5μlddH 2 O到50μl的最终体积。彻底混匀并短暂离心。
      2. 使用以下程序在热循环仪中运行:

    16. 用AMPure XP珠子进行DNA纯化
      1. 将150μl反相良好的AMPure XP珠加入到50μlDNA中,涡旋10秒,短暂离心并在室温孵育5分钟。
      2. 将试管放在磁力架上3分钟。
      3. 去除上清液。
      4. 加入200μl的80%乙醇,不打扰珠子,并在室温孵育30秒。去除EtOH。
      5. 重复洗涤步骤B16d。
      6. 将珠子风干5分钟。
      7. 从磁力架上取下管子。
      8. 加入22μl10mM Tris-HCl并上下移液以充分混合。在室温孵育2分钟。
      9. 将试管放在磁力架上3分钟。
      10. 将上清转移至1.5 ml微量离心管中。
    17. 用Qubit分析量化最终的文库浓度。
    18. 用Illumina双端测序法对RRBS文库进行测序。任何常规的双末端阅读长度,例如,100-250 bp,都可以。


  1. 我们的电子管 管道为每种酶及其相关的大小选择范围创建一张表格和条形图。图1是该流水线的流程图,用户可以看到每个步骤的输入和输出文件。图2是条形图,显示从大约40-300bp大小的选择的MseI消化的DNA中富集每个潜在的ROI。 X轴在log 2 范围内,所以值&gt; 0可被认为是这些Mse I消化的DNA片段的富集物。表1包括用于绘制图2的值。从图2和表1,我们可以看出,MseI消化的40-300bp片段富集于启动子中。作为替代方案,图3显示了另一种酶(CviQI)40-280bp的结果,其中片段在外显子,剪接位点,内含子和UTRs中更富集。
  2. 不同的酶产生相似的富集值。图2和图4表明在启动子中富含Mse I 40-300-bp和Bfa I 50-300-bp片段。表2和表3显示了所涵盖的启动子和基因体百分比的统计。对于MseI RR基因组,通过测序566Mbp的2.1Gb基因组,包括84.3%的启动子。至于BfaI RR基因组,测序23.0%的基因组包括74.8%的启动子。用户需要确定基因组4.4%和8.5%启动子的成本之间的权衡。


  1. 我们提供了一个计算机管道管道执行投资回报指导的RRBS在玉米与实验可验证的预测。用户运行我们管道的最关键步骤是:1)准备输入文件; 2)选择一种最佳的酶来显示所选ROI的清晰富集(见图2)。
  2. 输入文件通常可以从公共数据库下载,例如Ensembl(Zerbino et al。,2017)。需要注意的是,我们的管道需要一个单独的.gff3格式的重复注释文件。除了条形图和表格(图2和表3)外,当判断哪个大小选择区域是最佳的时候,还应该考虑测序平台的总RR基因组大小和阅读长度。例如,如果两个RR基因组在启动子中显示2个富集,一个覆盖整个基因组的50%,而另一个涵盖25%,则应该考虑测序成本和信息数量之间的折衷。
  3. 对于实验验证,尺寸选择和亚硫酸氢盐转化是最关键的步骤。在此协议中,我们使用凝胶大小选择来确保片段长度可以被可视化。作为替代方案,也可以使用AMPure XP珠子。为了确保有效的亚硫酸氢盐转化,可以在酶消化之前将0.01%剪切的λ噬菌体DNA汇集到原始基因组DNA中。由于λ噬菌体DNA缺乏甲基化胞嘧啶,通过计算λ噬菌体DNA的甲基化水平来评估亚硫酸氢盐转化效率可以估算出非转化率。


  1. 10 mM dNTP混合物
    10 mM dATP
    10 mM dTTP
    10 mM dCTP
    10 mM dGTP
  2. 5x RRBS dNTP混合


本研究得到中科院植物与微生物生物学研究所的资助和台湾科学技术部的资助(106-2633-B-001-001)至P.-Y.C.我们感谢Yi-Jing Lee提供设备信息。作者声明他们没有竞争性的经济利益。


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引用:Hsu, F., Wang, C. R. and Chen, P. (2018). Reduced Representation Bisulfite Sequencing in Maize. Bio-protocol 8(6): e2778. DOI: 10.21769/BioProtoc.2778.